Application of CCHC-type zinc finger protein PbrZFP719 gene in promoting pollen tube growth of pear

Abstract

The application discloses application of a CCHC type zinc finger protein PbrZFP719 gene in promoting pear pollen tube growth and belongs to the technical field of plant genetic engineering. The application clones the gene PbrZFP719 from 'yellow flower pear' pollen by using a plant gene cloning technique, the nucleotide sequence of the gene is shown in SEQ ID No. 1, and the amino acid sequence coded by the gene is shown in the sequence table SEQ ID No. 2. Research shows that the PbrZFP719 gene can promote pear pollen tube growth, the change of PbrZFP719 gene expression is related to the change of ROS level and cellulose content at the top of the pollen tube, and meanwhile, the PbrZFP719 gene also participates in the regulation mechanism of self-incompatibility reaction. The pollen magnetic transfection technology is used to study the function of the gene in the pollen tube, and provides a wide application prospect for improving pollination efficiency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant genetic engineering, and particularly relates to application of CCHC-type zinc finger protein (ZFP) PbrZFP719 gene in promoting growth of pear pollen tube. The present application clones CCHC-type ZFP PbrZFP719 gene from 'Huanghua' pollen, and the gene is highly expressed in pollen grains and pollen tubes. Through antisense oligonucleotide transfection (ODN) and pollen magnetic transfection, the PbrZFP719 gene is transiently knocked down or stably overexpressed in pear pollen tubes, and the results show that overexpression of the PbrZFP719 gene can promote the growth of pear pollen tubes. Further analysis shows that the change of PbrZFP719 gene expression is related to the change of reactive oxygen species (ROS) level and cellulose content at the top of the pollen tube. At the same time, the PbrZFP719 gene also participates in the self-incompatibility reaction of pear by responding to self S-RNase. BACKGROUND

[0002] Self-incompatibility (SI) is a reproductive strategy to prevent self-pollination and promote cross-pollination to improve species diversity. Rosaceae fruit trees, such as pear and apple, exhibit gametophytic self-incompatibility (GSI). GSI is controlled by S-loci containing female determinant S-RNase and male determinant SLFs / SFBs (Franklin et al. 2008; Kao et al. 2004; Chen et al. 2018; Ushijima K et al. 2003). When pollen from the same S-locus falls on the stigma, S-RNase produces toxicity and destroys cell structure, such as destroying cell wall structure and affecting ROS level, ultimately leading to fertilization failure (Wang et al. 2010; Chen et al. 2018; Wu et al. 2023a). In contrast, pollen from different S-loci fertilizes, and SLFs / SFBs genes of Skp1-Culin1-F-box (SCF) complex can neutralize S-RNase, promoting pollen tube elongation (Hua et al. 2006). Currently, factors outside S-loci related to SI response have also been found, including leucine-rich repeat extension protein (LRX; Wu et al. 2023a), GPI-anchored protein (COBRA; Wu et al. 2023b), myo-inositol oxygenase (MIOX3; Xu et al. 2024), pectin methylesterase (PME44; Tang et al. 2023), self-incompatibility pollen protein (SIPP; Garcia Valencia et al. 2017), and soluble inorganic pyrophosphatase (PPa; Li et al. 2018).

[0003] Zinc finger proteins (ZFPs) contain at least one zinc finger motif, including C4HC3, C3H, C3HC4, CCHC, C2H2, C2HC5, C8, C4 and C6 types (Berg and Shi 1996). CCHC type ZFPs contain the conserved amino acid sequence feature Cys-X2-Cys-X4-His-X4-Cys. These proteins have RNA or single-stranded DNA binding activity and are involved in protein-protein interactions (Liew et al. 2000). Therefore, CCHC type ZFPs may play a role in transcriptional regulation, translational regulation and pre-mRNA splicing. However, whether CCHC type ZFPs are involved in pollen tube growth and self-incompatibility reactions remains to be explored.

[0004] Pears are a fruit that is deeply loved by the people, and successful pollination and fertilization is the key to ensuring pear yield. The present application obtains a PbrZFP719 gene highly expressed in pear pollen, and studies the effect of the PbrZFP719 gene on pear pollen tube growth and the role played by the PbrZFP719 gene in the process of pear self-incompatibility. Exploring and applying pollen magnetic transfection technology to practice can greatly reduce the cost of artificial pollination, and has considerable theoretical and practical significance for agricultural production. SUMMARY

[0005] The purpose of the present application is to provide the application of PbrZFP719 gene or biological material related to the PbrZFP719 gene in promoting pear pollen tube growth. The applicant screens a PbrZFP719 gene highly expressed in pollen tube from 'yellow flower pear', and the PbrZFP719 gene belongs to the CCHC type ZFPs family gene. The pollen magnetic transfection technology is applied to the study of pollen overexpression, and combined with pollen ODN transfection, it is identified that the PbrZFP719 gene has the function of affecting the ROS level and cellulose content of the pollen tube tip and affecting the pear pollen tube growth.

[0006] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0007] In a first aspect, the present application claims the application of PbrZFP719 gene or biological material related to the PbrZFP719 gene in at least one of the following (a1)-(a8):

[0008] (a1) the application in promoting pear pollen tube growth;

[0009] (a2) the application in preparing a product for promoting pear pollen tube growth;

[0010] (a3) the application in improving the ROS level of the tip of pear pollen tube;

[0011] (a4) the application in preparing a product for improving the ROS level of the tip of pear pollen tube;

[0012] (a5) use in increasing the cellulose content of the pollen tube tip of Pyrus;

[0013] (a6) use in the preparation of a product for increasing the cellulose content of the pollen tube tip of Pyrus;

[0014] (a7) use in increasing the efficiency of in vitro pollination of Pyrus;

[0015] (a8) use in the preparation of a product for increasing the efficiency of in vitro pollination of Pyrus;

[0016] The PbrZFP719 gene is any one of the following (b1)-(b4) DNA molecules:

[0017] (b1) a DNA molecule whose coding region comprises the nucleotide sequence shown in SEQ ID NO. 1;

[0018] (b2) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO. 1;

[0019] (b3) a DNA molecule which hybridizes to the DNA sequence defined in (b1) or (b2) under stringent conditions and encodes a protein associated with promoting pollen tube growth of Pyrus.

[0020] Further, the biological material associated with the PbrZFP719 gene is at least one of the following (c1)-(c6):

[0021] (c1) a protein encoded by the PbrZFP719 gene;

[0022] (c2) an expression cassette containing the PbrZFP719 gene;

[0023] (c3) a recombinant vector containing the PbrZFP719 gene, or a recombinant vector containing the expression cassette of (c2);

[0024] (c4) a recombinant microorganism containing the PbrZFP719 gene, or a recombinant microorganism containing the expression cassette of (c2), or a recombinant microorganism containing the recombinant vector of (c3);

[0025] (c5) a transgenic plant cell line containing the PbrZFP719 gene, or a transgenic plant cell line containing the expression cassette of (c2), or a transgenic plant cell line containing the recombinant vector of (c3);

[0026] (c6) a magnetofection reagent containing the PbrZFP719 gene, or a magnetofection reagent containing the expression cassette of (c2), or a magnetofection reagent containing the recombinant vector of (c3).

[0027] Still further, the protein encoded by the PbrZFP719 gene is as follows (d1) or (d2) or (d3):

[0028] (d1) a protein having an amino acid sequence as shown in SEQ ID NO. 2;

[0029] (d2) a protein derived from SEQ ID NO. 2 by substitution and / or deletion and / or addition of one or several amino acid residues and having a function of promoting pollen tube growth of Pyrus;

[0030] (d3) a fusion protein of (d1) or (d2) with a protein tag connected to the N-terminus or / and C-terminus.

[0031] Further, the application is to stably overexpress the PbrZFP719 gene in pollen tubes of Pyrus, promote pollen tube growth of Pyrus, or / and increase the ROS level or / and cellulose content at the tip of pollen tubes of Pyrus. Still further, the PbrZFP719 gene is stably overexpressed in pollen tubes of Pyrus by using magnetic transfection overexpression technology to treat pollen of Pyrus in vitro.

[0032] The applicant screened a PbrZFP719 gene overexpressed in pollen tubes from Pyrus, which belongs to the CCHC-type ZFPs family gene; and cloned the CCHC-type ZFPs family gene PbrZFP719 from Pyrus by using gene cloning technology. The PbrZFP719 nucleotide sequence is shown in SEQ ID No. 1, which contains an open reading window of 843 bp, encodes 281 amino acids, the encoded amino acid sequence is shown in SEQ ID No. 2, the isoelectric point is 9.55, and the molecular weight is 30.96 kDa.

[0033] The applicant designed a primer pair for cloning the cDNA sequence of the above-mentioned gene PbrZFP719, and the base sequence is as follows:

[0034] PbrZFP719 F1: 5'-ATGGCAGGGAAGGAAGACTACC-3' (SEQ ID No. 3)

[0035] PbrZFP719 R1: 5'-GTAACGGTCAAAGGAAGATGGG-3' (SEQ ID No. 4).

[0036] The ODN and magnetic transfection overexpression technology are used to treat pollen of Pyrus in vitro, and it is verified that PbrZFP719 has the functions of promoting pollen growth, affecting the ROS level at the tip of pollen tubes and the cellulose content.

[0037] In a second aspect, the present application claims a method for promoting growth of pear pollen tube, stably overexpressing the PbrZFP719 gene in the pear pollen tube to promote growth of the pear pollen tube.

[0038] In a third aspect, the present application claims a method for increasing ROS level and cellulose content at the tip of pear pollen tube, stably overexpressing the PbrZFP719 gene in the pear pollen tube to increase ROS level and / or cellulose content at the tip of the pear pollen tube.

[0039] In a fourth aspect, the present application claims a method for improving efficiency of in vitro pollination of pear, stably overexpressing the PbrZFP719 gene in the pear pollen tube to promote growth of the pear pollen tube and improve efficiency of in vitro pollination of the pear.

[0040] Further, in the above method, the PbrZFP719 gene is stably overexpressed in the pear pollen tube by using magnetic transfection overexpression technology to treat the pear pollen in vitro.

[0041] (1) designing primers to PCR-amplify the PbrZFP719 gene, inserting the PbrZFP719 gene into the enzyme cleavage sites of XbaI and BamHI of the LAT52::GFP vector to construct a recombinant plasmid PbrZFP719-LAT52::GFP;

[0042] (2) mixing the transfection reagent and the recombinant plasmid PbrZFP719-LAT52::GFP to prepare / PbrZFP719-LAT52::GFP transfection reagent, and using the / PbrZFP719-LAT52::GFP transfection reagent to treat the pollen cells.

[0043] The present application uses plant gene cloning technology to clone the gene PbrZFP719 from the pollen of ‘Yellow Flower Pear’, which belongs to the CCHC type ZFPs gene family member and is highly expressed in pollen grains and pollen tubes. The ‘Yellow Flower Pear’ pollen is treated in vitro by ODN and pollen magnetic transfection test, and the results show that overexpression of the gene PbrZFP719 can actively promote the growth of pollen tubes, and the change of PbrZFP719 expression is related to the change of ROS level and cellulose content at the tip of the pollen tube. In addition, PbrZFP719 is also involved in the regulation mechanism of self-incompatibility reaction. The present application uses pollen magnetic transfection technology to study the function of genes in pollen tubes, which provides a broad application prospect for improving pollination efficiency.

[0044] The room temperature in the present application is generally 25±10℃, but is not limited thereto. ​

[0045] Compared with the prior art, the application has advantages and effects:

[0046] (1) The discovery of the PbrZFP719 gene provides a new idea for improving the in vitro pollination efficiency of pears, reduces labor costs, and provides a new way for implementing green agriculture.

[0047] (2) Compared with the traditional pollen gene gun technology, the method of overexpressing pollen tube genes through pollen magnetic transfection technology has the advantages of high transformation efficiency, simple operation, cost saving, and directional improvement of traits. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 For identification of CCHC type ZFPs family members in pears.

[0049] Figure 2 For the transcriptome expression pattern of 14 CCHC type ZFPs genes in different tissues.

[0050] Figure 3 For the PbrZFP719 gene positively promoting pollen tube growth;

[0051] Wherein, A is the phenotype image of as-ODN, s-ODN, cell transfection and control treatment of pollen tube, and the black line represents a scale (50 μm); B is the length of pollen tube treated by as-ODN, s-ODN, cell transfection and control; C is the expression level of PbrZFP719 gene in pollen tube treated by different treatments.

[0052] Figure 4 For the application of magnetic transfection of PbrZFP719 gene in promoting pollen tube growth;

[0053] Wherein, A is the fluorescence picture of pollen magnetic transfection. The red line represents a scale (10 μm); B is the phenotype image of pollen tube transferred by PbrZFP719-LAT52::GFP fusion vector and LAT52::GFP vector, and the red line represents a scale (50 μm); C is the length of pollen tube determined by pollen magnetic staining experiment; D is the expression level of PbrZFP719 in pollen tube detected by pollen magnetic staining experiment.

[0054] Figure 5 For the PbrZFP719 gene positively mediating the ROS level and cellulose content of the tip of pollen tube;

[0055] Wherein, A is the fluorescence value of ROS in the pollen tube treated with as-ODN, s-ODN, cell transfection and control by CM-H2DCFDA staining; B is the ROS level in the pollen tube detected by pollen magnetic staining experiment; C is the fluorescence value of cellulose in the pollen tube treated with as-ODN, s-ODN, cell transfection and control; D is the image of the cellulose content in the pollen tube induced by lat52::GFP vector and PbrZFP719-lat52::GFP fusion vector.

[0056] Figure 6 Self S-RNase reduces the expression of PbrZFP719 gene;

[0057] Wherein, A is the ROS level in the pollen tube treated with self S-RNase, non-self S-RNase and control; B is the fluorescence value of cellulose in the pollen tube after treatment; C is the expression level of PbrZFP719 in the pollen tube treated with self S-RNase, non-self S-RNase and control. Specific embodiments

[0058] The present application is described in detail below in conjunction with specific embodiments. Based on the following description and these embodiments, those skilled in the art can determine the essential features of the present application, and make various changes and modifications to the present application without departing from the spirit and scope of the present application, so as to adapt the present application to various uses and conditions.

[0059] Example 1 Tissue localization of PbrZFP719 gene

[0060] CCHC-ZFP type genes were identified from the genome of pear (http: / / www.peargenome.njau.edu.cn) using hidden Markov model (PF00098). The isoelectric point and molecular weight were calculated using ExPasy (http: / / web.expasy.org / protparam / ). The conserved motifs shared by pear gene family members were searched in MEME (http: / / meme-suite.org / tools / meme) using the amino acid sequence. A total of 14 CCHC type ZFP genes were identified in pear, all of which contain a CCHC domain (as shown in Figure 1

[0061] ​The transcriptome data from previous studies were used to elucidate the expression patterns of 14 CCHC-type ZFPs genes in various tissues. Transcriptome analysis revealed the expression patterns of 14 CCHC-type ZFPs genes in pollen tube, inflorescence, petiole, petiole and leaf. S1 to S6 represent 30, 45, 60, 75, 90 and 110 days after harvest. 0, 1, 6 and 15h represent the time of pollen incubation in the medium. The results show that PbrZFP719 exhibits high level of expression in all the developmental stages of the tissues tested (as shown in Figure 2 Figure 1), especially in pollen grains and pollen tubes, suggesting its potential role in pollen tube growth.

[0062] Example 2 Identification of PbrZFP719 on pollen tube growth

[0063] The antisense oligodeoxynucleotide sequence (as-ODN) and the sense oligonucleotide sequence (s-ODN) of PbrZFP719 were designed by the RNAfold website (http: / / rna.tbi.univie.ac.at). At the same time, the primer sequences were modified by thio and purified by HPLC.

[0064] The PbrZFP719-ODN primers are as follows:

[0065] PbrZFP719-as-ODN: 5'-CGCCTCTAGCTGGCCCTCCGC-3' (SEQ ID No. 5)

[0066] PbrZFP719-s-ODN: 5'-GCTATGGAGAGAGGGACCGT-3' (SEQ ID No. 6)

[0067] The specific steps are as follows: an appropriate amount of pear pollen was added to 2 mL of medium (5 mM 2-morpholinoethanesulfonic acid (MES), 440 mM sucrose, 0.55 mM calcium nitrate, 1.60 mM magnesium sulfate, 1.60 mM boric acid, 1.00 mM potassium nitrate, pH = 6.2-6.3) and incubated on a shaker for 60 min to obtain the incubated pollen medium. 12.5 μL of the medium, 1.5 μL of Lipofectamine 2000 and 6 μL of ODN primer were incubated at room temperature for 15 min to obtain the premixed ODN primer; the premixed ODN primer was added to 180 μL of the incubated pollen medium; the pollen was further incubated at 25°C for 3 h. Nikon Eclipse E100 microscope (Tokyo, Japan) was used for observation and photography, and Image J was used to measure the pollen tube length (as shown in Figure 3A) and (B) in FIG. 3. The results show that after 3 hours of treatment (HAT), the pollen tubes treated with as-ODN were shorter than those treated with s-ODN, transfection reagent and medium buffer (e.g. Figure 3 A) and (B) in FIG. 3. The results show that after 3 hours of treatment (HAT), the pollen tubes treated with as-ODN were shorter than those treated with s-ODN, transfection reagent and medium buffer (e.g.

[0068] 3000 rpm, 10 min to collect the pollen, remove the liquid medium, and store in a -80°C refrigerator. Extract the pollen RNA, reverse transcribe to obtain the first strand cDNA, and use it for qRT-PCR experiment of PbrZFP719 gene. The RNA extraction uses a plant total RNA extraction kit (purchased from Beijing Tiangen Biotech Co., Ltd., and operated according to the operation instruction provided by the kit). The RNA reverse transcription to cDNA uses TransScript One-Step RT-PCR SuperMix (purchased from Beijing Zonnuo Biotech Co., Ltd., and operated according to the instruction provided by the kit). The specific quantitative primers of PbrZFP719 gene are designed as follows:

[0069] PbrZFP719-qPCR-F: 5'-ATGATAGGGATGGTGGCGCAA-3' (SEQ ID No. 7)

[0070] PbrZFP719-qPCR-R: 5'-AACGGTCAAAGGAAGATGGGC-3' (SEQ ID No. 8)

[0071] The specific quantitative primers of pear UBQ gene as the internal reference are as follows:

[0072] PbUBQ-F: 5'-CCCTTCACTTGGTTCTCCGT-3' (SEQ ID No. 9)

[0073] PbUBQ-R: 5'-TAATCAGCAAGCGTGCGACC-3' (SEQ ID No. 10)

[0074] qRT-PCR experiments were performed using the LC480 SYBR Green Mix kit (purchased from Roche) according to the kit instructions. The 20 μL qRT-PCR reaction system included: 10 μL 2x SYBR Green Mix, 0.4 uM forward and reverse primers, 20 ng cDNA, and the rest was supplemented with sterile water. The 96-well qRT-PCR plate (purchased from Roche) was used, and the qRT-PCR instrument (model: LightCycler 480, Roche) was used for PCR. The qRT-PCR reaction program was: 95°C pre-denaturation for 10 min; 95°C denaturation for 3 s, 62°C annealing for 10 s, 72°C extension for 30 s, 45 cycles. Each cDNA was set up with three biological replicates and three technical replicates, and the average Ct value of each cDNA sample was calculated. The relative expression of each gene was calculated by 2 -ΔΔCt The relative expression of PbrZFP719 gene was obtained. The standard error was based on 3 repeated qRT-PCR analysis and at least 90 pollen tube growth analysis. The variance analysis used Student's t-test, and the letters (a and b) represented P<0.05. As shown in C of Figure 3 The expression level of PbrZFP719 gene in as-ODN treated pollen tube was decreased. These results showed that the decrease of PbrZFP719 expression inhibited the growth of pear pollen tube.

[0075] Example 3 Application of magnetic transfection of PbrZFP719 gene in promoting pollen tube growth

[0076] In the present application, the plasmid vector is LAT52::GFP vector (Qian et al. 2020). The resistance tag of this vector is kanamycin, and the double enzyme digestion is preferably XbaI and BamHI (purchased from NEB). The optimal reaction system and conditions of enzyme digestion: 800 ng LAT52::GFP empty plasmid, 5 μL 10x Cutsmart Buffer, 1 μL BamHI endonuclease, 1 μL XbaI endonuclease, supplemented with ddH2O to 50 μL, 37°C reaction for 4 h.

[0077] The primer pair for PCR amplification of the gene is:

[0078] PbrZFP719-F1: 5'-ATGGCAGGGAAGGAAGACTACC-3' (SEQ ID No. 3)

[0079] PbrZFP719-R1: 5'-GTAACGGTCAAAGGAAGATGGG-3' (SEQ ID No. 4)

[0080] PCR amplification system: 2 μL 'yellow flower pear' pollen cDNA, 2.5 μL of each primer, 25 μL 2x PhantaMax Buffer, 1 μL dNTP Mix, 1 ul PhantaMax Super-Fidelity DNA Polymerase (purchased from Nanjing Novozyme Biotech Co., Ltd.), and ddH2O to 50 μL. The amplification program is: 94℃, pre-denaturation 3min, 94℃ denaturation 30s, 60℃ annealing 30s, 72℃ extension 140s, 35 cycles, 72℃ extension 10min, 4℃ preservation. After amplification, the PCR product with a single band of interest was detected by 1.5% agarose gel electrophoresis, and the specific band was recovered according to the gel recovery kit (purchased from Nanjing Novozyme Biotech Co., Ltd.).

[0081] The double enzyme-digested vector was ligated with the purified DNA, and the ligase Exnase II was purchased from Nanjing Novozyme Biotech Co., Ltd. The reaction system was 20 μL: 150 ng LAT52::GFP linear vector, 50 ng gene fragment, 4 μL 5x CE II Buffer, 2 μL Exnase II, and the rest was supplemented with ddH2O, 37℃ for 30min. Then the ligation product was transferred into E. coli DH5a (purchased from Nanjing Novozyme Biotech Co., Ltd.), ice bath for 30min, heat shock for 45s, then ice bath for 2min, 37℃ 220rpm shaking bed activation for 60min. After activation, it was coated on the LB solid plate with 100 μg / ml kanamycin, and after 14 hours, 5 positive clones were picked for sequencing (completed by Shanghai Sunway Biotech Co., Ltd.). The recombinant plasmid PbrZFP719-LAT52::GFP with successful sequencing was extracted using endotoxin-free plasmid large extraction kit (purchased from Nanjing Novozyme Biotech Co., Ltd.).

[0082] The optimal method of pollen magnetic transfection is as follows: put 'yellow flower pear' pollen cells into a tissue culture dish placed on a magnetic marker plate (purchased from Nanjing Dongna Co., Ltd.), and the volume of the medium containing cells depends on the specification of the culture dish, preferably 0.5mL transfection volume, incubate for 15min. Mix the transfection reagent (purchased from Nanjing Dongna Co., Ltd.) and PbrZFP719-LAT52::GFP recombinant plasmid, preferably 0.5-2 μL, DNA amount 0.5-3 μg. LAT52::GFP empty plasmid as control; the prepared The transfection reagent and control reagent were added into the cells, respectively, and the cell culture dishes were still placed on the magnetic marker plate for incubation for 20 minutes; the supernatant pollen culture medium was carefully removed from the cells, and fresh culture medium was added, and the culture plate was still placed on the magnetic marker plate. Care should be taken not to suck away the cells sinking due to magnetic force; the culture plate was removed from the magnetic marker plate; after 3h standard condition culture at 25℃ and 120rpm, whether the GFP fluorescence existed in the magnetic transfection pollen was observed by laser confocal microscope LSM800 (Zeiss, Germany), and whether the MNP / DNA complex was transferred into the pollen was determined. The pollen tube length was counted by Nikon Eclipse E100 microscope (Tokyo, Japan), and data analysis was performed. The RNA of the pollen after magnetic transfection was extracted, and the first strand cDNA was obtained by reverse transcription, which was used for qRT-PCR experiment of PbrZFP719 gene. The pollen RNA extraction, cDNA reverse transcription and qRT-PCR experiment were the same as those in Example 2. The standard error was based on the qRT-PCR analysis of 3 times repetition and at least 90 pollen tube growth analysis. The variance analysis used Student's t test, and the letters (a and b) represented P < 0.05.

[0083] The results showed that the GFP fluorescence was detected in the pollen tube with the introduction of LAT52::GFP or PbrZFP719-LAT52::GFP fusion vector, which indicated the applicability of pollen magnetic staining technology in the research of gene overexpression in pear pollen tube (as shown in A of Figure 4 The three HATs (as shown in B and C of Figure 4 The pollen tube with the introduction of PbrZFP719-LAT52::GFP fusion vector was longer than that with the introduction of LAT52::GFP vector. At the same time, compared with the LAT52::GFP vector, the expression amount of PbrZFP719 in the pollen tube with the PbrZFP719 fusion vector was increased (as shown in D of Figure 4 These results showed that the increase of PbrZFP719 expression amount promoted the growth of pear pollen tube.

[0084] Example 4 Identification of PbrZFP719 affecting the content of ROS and cellulose in pollen tube

[0085] The pollen culture was the same as that in Examples 2 and 3.

[0086] The present application uses H2DCFDA (Thermo Fisher Scientific, USA) fluorescent staining method to determine the ROS level of pollen tube tip, the optimal method is as follows: after adding H2DCFDA fluorescent dye with a final concentration of 20 mM to the pollen sample for 20 min, the sample is washed with liquid medium for three times; laser confocal microscope LSM800 (Zeiss, Germany) is used for observation and photography, and Zeiss software is used to count the fluorescence intensity of pollen tube tip. Calcofluor White (Merck, Germany) is used for cellulose staining of pollen tube, and the final concentration is 1 pg / mL.

[0087] The results show that the ROS fluorescence of pollen tube tip treated by as-ODN decreases compared with s-ODN, transfection reagent and buffer treated pollen tube (as shown in A of Figure 5 In contrast, the pollen tube tip introduced with PbrZFP719-LAT52::GFP vector shows higher ROS fluorescence compared with LAT52::GFP vector (as shown in B of Figure 5 These results show that PbrZFP719 gene plays a positive role in the ROS level of pear pollen tube tip. In addition, Calcofluor White fluorescent staining shows that the cellulose fluorescence decreases after as-ODN treatment (as shown in C of Figure 5 and the cellulose fluorescence of pollen tube introduced with PbrZFP719-LAT52::GFP is enhanced (as shown in D of Figure 5 These results show that PbrZFP719 gene also positively mediates the cellulose content of pear pollen tube tip.

[0088] Example 5 Identification of PbrZFP719 gene involved in self-incompatibility reaction

[0089] The full-length coding sequences of PbrS1- and PbrS2-RNase genes are amplified from the style of‘Yellow Flower Pear’, and the full-length coding sequences of PbrS7- and PbrS34-RNase genes are amplified from the style of‘Dangshan Pear’; four S-RNases are inserted into pCold-TF expression vector to produce his-labeled recombinant protein; BamHI and XbaI are selected as endonucleases. The primer sequences are as follows:

[0090] PbrS1-RNase-F: 5’-ATGTACGATTATTTTCAATTTACGCAGCAAT-3’ (SEQ ID No. 11) PbrS1-RNase-R: 5’-ATACTGAACACTGGAGGGGCAGG-3’ (SEQ ID No. 12)

[0091] PbrS2-RNase-F: 5'-ATGGCGAGATACGATTATTTTCAATTTACGC-3' (SEQ ID No. 13) PbrS2-RNase-R: 5'-ATACTGAATATCATCAATGGGGCAGAA-3' (SEQ ID No. 14)

[0092] PbrS7-RNase-F: 5'-ATGTACGATTATTTTCAATTTACGCAGCAAT-3' (SEQ ID No. 15) PbrS7-RNase-R: 5'-ATACTTAACATCGGCCGGGCAG-3' (SEQ ID No. 16)

[0093] PbrS34-RNase-F: 5'-ATGTACGATTATTTTCAATTTACGCAGCAAT-3' (SEQ ID No. 17) PbrS34-RNase-R: 5'-ATACTGAATACTATTGTTTGGGCAAAAATG-3' (SEQ ID No. 18)

[0094] The preferred procedure of PCR, enzyme digestion system and recombination vector construction are the same as those in Example 3.

[0095] 500 ng of the recombination plasmid was transformed into E. coli Rosetta (DE3) respectively, and spread on LB plate containing 100 μg / mL ampicillin to screen the recombination gene, and cultured in 37 °C incubator for 14 h. The pCold-TF vector plasmid was also transformed into Rosetta (DE3) as a control. Single colony was selected for identification. The E. coli Rosetta (DE3) transformed with the recombination plasmid was inoculated in LB liquid screening medium for activation, and cultured at 37 °C, 220 rpm overnight, then transferred to new liquid screening medium for culture with OD 600 = 0.4-0.6, and the inoculation amount of the activation culture and the expansion culture was preferably 1:50 by volume ratio; 2 ml of the bacterial solution was taken as a negative control. The expansion culture conical flask was quickly placed on ice for 45 min, then IPTG inducer was added, preferably with a final concentration of 0.5 mmol / L, and induced to express at 16 °C, 220 rpm for 18-24 h.

[0096] After expression, the bacteria were collected by centrifugation at 4°C, 12000 rpm. The bacteria were resuspended in 15 mL PBS buffer (140 mmol / L NaCl, 2.7 mmol / L KCl, 10 mmol / L Na2HPO4, 1.8 mmol / L KH2PO4, pH 7.4) and then broken by ultrasonication at 240 W, 4 s on and 6 s off until the solution was clear. After ultrasonication, the supernatant was collected by centrifugation at 4°C, 12000 rpm for 20 min. The control protein pCold-TF was also expressed by the above method.

[0097] The recombinant protein was purified by Ni-NTA agarose affinity chromatography (purchased from Shanghai Gener Biotech Co., Ltd.). Specifically, the above-mentioned filler was equilibrated with 10 times the volume of PBS buffer, and the flow rate was controlled at 1 ml / min. The supernatant of the broken protein was added to the purification column, and the flow rate was controlled at 0.5 ml / min. The column was washed with 20 times the volume of washing solution containing 20 mmol / L imidazole (500 mmol / L NaCl, 50 mmol / L Tris (hydroxymethyl) aminomethane, 20 mM imidazole, pH = 7.4), and the flow rate was controlled at 1 ml / min. The purification column was eluted with 8 times the volume of elution solution containing 400 mmol / L imidazole (500 mmol / L NaCl, 50 mmol / L Tris (hydroxymethyl) aminomethane, 400 mM imidazole, pH = 7.9), and the flow rate was controlled at 1 ml / min. The purified protein was collected by collecting the eluate. Protein concentration and desalination were performed using a 30 kDa ultrafiltration tube. 10 μL of the purified protein was taken, 2 μL of 5x protein loading buffer (purchased from Shanghai Gener Biotech Co., Ltd.) was added, and 10 μL was taken for 12% regular SDS-PAGE electrophoresis. After staining with Coomassie brilliant blue and decolorizing, the purification of the recombinant protein was detected. Finally, the purified protein was dialyzed at 4°C, 5000 rpm using pollen culture medium, and then stored at -80°C.

[0098] The "yellow rosewood" pollen was pre-cultured in the basic medium for 1 h, and then treated with recombinant PbrS1- and PbrS2-RNase proteins or recombinant PbrS7- and PbrS34-RNase proteins (final concentration of 0.15 U). Then, it was cultured at 25°C, 120 rpm for 30 min on a shaker. Part of the pollen was stained with H2DCFDA and Calcofluor White for pollen tube tip staining, and the steps were the same as in Example 4. The remaining pollen was extracted for RNA, cDNA reverse transcription, and qRT-PCR experiments, which were the same as in Example 2.

[0099] To verify whether PbrZFP719 gene was involved in GSI, we treated pollen tubes with self (PbrS1- and PbrS2-RNase) and non-self S-RNases (PbrS7- and PbrS34-RNase) for 30 min. The results showed that the ROS and cellulose fluorescence of the pollen tube tips treated with self SI were lower than those treated with non-self SC and the control group pCold (as shown in A and B of FIG. 6). Real-time fluorescent quantitative PCR analysis showed that the expression level of PbrZFP719 in the pollen tubes treated with self SI was lower than that treated with non-self SC and pCold (as shown in C of FIG. 6). Therefore, PbrZFP719 gene was involved in pear GSI by responding to self S-RNase. Figure 6 Figure 6

[0100] The above description is only preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

[0101] SEQ ID NO: 1

[0102] ​​ATGGCAGGGAAGGAAGACTACCGTATATTCGTGGGAGGGTTGTCGTGGGACGTGACGGAGCGTCAGCTGGAGAGCGCTTTCCAACGCTTCGGCAAAGTTCTTGAAGCTCAGATCATGATGGAAAGAGATACAAACCGTCCCCGAGGATTTGGGTTTATTACATTTGGAGATCGGCGAGCAATGGAAGACGCAATCCGGGAGATGCATGGGCGGGAGCTTAGTGATCGCATAATCTCTGTGAACAAAGCTCAACCCAAAATGGGAGGGGGAGGAGCTGGAGAGGATTCTGACCATGGCTACAGAGGAGGTGGCTACTCGTCTGGTGGCAGGAGAAACTATGGGGGAGGAGATAGACCTGTAGGACAAGATGAGTGCTTCAAGTGTGGTCGAACAGGACATTGGGCTCGAGATTGCCCTTCAGCAGGAGGCGGAAGAGGCGGTGGAGGTTCATTCTCATCACATTCTAGGTTTGGGGCTGGTGGCCGTGGGGATCGCTTTGGTGGAGACCGTGACCGCTACATGGATGACCGTTATGATGGAGGGCGCTATGGAGAGAGGGACCGTTTTGACAGCAGAGATGACAAATATGGTAGCCGTGATCGCTATGCTAGCGACAGGTACCCAGCCGGTGATCGTTTTGCAAGTGACAGGTACGGTGGTTCTGATCGTTATCCTCAAAATGGTTATGGCAAAGATAGAGGGTATGATAGGGATGGTGGCGCAAGAGGAGGCGACAGGTATGCAAGCGGAGGGCCAGCTAGAGGCGATAATTACAGAAGCAGGCCTGGTCCTTATGACCGCCCTAGCAGGGGAGGCCGCCCATCTTCCTTTGACCGTTACTAA

[0103] SEQ ID NO: 2

[0104] MAGKEDYRIFVGGLSWDVTERQLESAFQRFGKVLEAQIMMERDTNRPRGFGFITFGDRRAMED AIREMHGRELSDRIISVNKAQPKMGGGGAGEDSDHGYRGGGYSSGGRRNYGGGDRPVGQDE CFKCGRTGHWARDCPSAGGGRGGGGSFSSHSRFGAGGRGDRFGGDRDRYMDDRYDGGRY GE RDRFDSRDDKYGSRDRYASDRYPAGDRFASDRYGGSDRYPQNGYGKDRGYDRDGGARG GD RYASGGPARGDNYRSRPGPYDRPSRGGRPSSFDRY*.

Claims

1. Use of a nucleotide sequence as shown in SEQ ID NO. 1 PbrZFP719 the gene in at least one of the following (al) - (a8): (a1) use in promoting pear pollen tube growth; (a2) use in the preparation of a product for promoting pear pollen tube growth; (a3) use in increasing the level of ROS at the tip of pear pollen tube; (a4) use in the preparation of a product for increasing the level of ROS at the tip of pear pollen tube; (a5) use in increasing the cellulose content at the tip of pear pollen tube; (a6) use in the preparation of a product for increasing the cellulose content at the tip of pear pollen tube; (a7) use in increasing the efficiency of pear in vitro pollination; (a8) use in the preparation of a product for increasing the efficiency of pear in vitro pollination.

2. A biological material associated with a nucleotide sequence as set forth in SEQ ID NO. 1 PbrZFP719 application of the biological material associated with the gene in at least one of (al)-(a8) below: (a1) use in promoting pear pollen tube growth; (a2) use in the preparation of a product for promoting pear pollen tube growth; (a3) use in increasing the level of ROS at the tip of pear pollen tube; (a4) use in the preparation of a product for increasing the level of ROS at the tip of pear pollen tube; (a5) use in increasing the cellulose content at the tip of pear pollen tube; (a6) use in the preparation of a product for increasing the cellulose content at the tip of pear pollen tube; (a7) use in increasing the efficiency of pear in vitro pollination; (a8) use in the preparation of a product for increasing the efficiency of pear in vitro pollination. The above and PbrZFP719 Gene-related biological materials are at least one of the following (c1)-(c6): (c1 ) the protein encoded by the gene PbrZFP719 gene; (c2) a vector comprising the expression cassette of (c1); and PbrZFP719 a gene; (c3) a recombinant vector comprising the nucleic acid molecule of (c2) or (c1 ), or a recombinant vector comprising the expression cassette of (c2); and PbrZFP719 (c3) a recombinant vector comprising the nucleic acid molecule of (c2) or (c1 ), or a recombinant vector comprising the expression cassette of ( (c4) a recombinant microorganism comprising the PbrZFP719 gene, or a recombinant microorganism comprising the expression cassette of (c2), or a recombinant microorganism comprising the recombinant vector of (c3); (c5) a transgenic plant cell line comprising said PbrZFP719 a transgenic plant cell line comprising (c2) said expression cassette, or a transgenic plant cell line comprising (c3) said recombinant vector; (c6) a magnetic transfection reagent containing said PbrZFP719 gene, or a magnetic transfection reagent containing (c2) said expression cassette, or a magnetic transfection reagent containing (c3) said recombinant vector.

3. Use according to claim 2, characterized in that, The PbrZFP719 The protein encoded by the gene is a protein with an amino acid sequence as shown in SEQ ID NO.

2.

4. Use according to claim 1, characterized in that, The method comprises the following steps: PbrZFP719 The gene is stably overexpressed in pear pollen tubes, promotes pear pollen tube growth, or / and increases the ROS level or / and the cellulose content of the top of the pear pollen tube.

5. Use according to claim 4, characterized in that, The gene was stably overexpressed in pear pollen tubes by using magnetic transfection overexpression technology to treat pear pollen in vitro. PbrZFP719 The gene was stably overexpressed in pear pollen tubes by using magnetic transfection overexpression technology to treat pear pollen in vitro.

6. A method of promoting pollen tube growth in pear, characterized by, The method of claim 1, wherein the compound is PbrZFP719 Stable overexpression of the gene in pear pollen tubes promotes pear pollen tube growth.

7. A method of increasing the level of ROS or / and the content of cellulose at the pollen tube tip of Pyrus, characterized in that, The method of claim 1, wherein the compound is PbrZFP719 The gene is stably overexpressed in pear pollen tube, and the ROS level or / and the cellulose content at the top of the pear pollen tube is increased.

8. A method of increasing the efficiency of pollination of a pear in vitro, characterized in that, The method of claim 1, wherein the compound is PbrZFP719 The gene is stably overexpressed in pear pollen tubes, promotes pear pollen tube growth, and improves pear in vitro pollination efficiency.

9. The method according to any one of claims 6-8, characterized in that, The gene was stably overexpressed in pear pollen tubes by using magnetic transfection overexpression technology to treat pear pollen in vitro. PbrZFP719 The gene was stably overexpressed in pear pollen tubes by using magnetic transfection overexpression technology to treat pear pollen in vitro.

10. The method of claim 9, wherein, The magnetic transfection overexpression technology specifically comprises the following steps: (1) Design primers for PCR amplification PbrZFP719 genes, insert the PbrZFP719 genes into the Xbal and BamHI restriction sites of the LAT52::GFP vector to construct recombinant plasmid PbrZFP719 - LAT52::GFP; (2) The MagTransf® transfection reagent was mixed with the recombinant plasmid PbrZFP719-LAT52::GFP to make MagTransf® / PbrZFP719 - The MagTransf® / PbrZFP719 transfection reagent was used to transfect pollen cells. - The MagTransf® / PbrZFP719 transfection reagent was used to transfect pollen cells.

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